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During chronic infection, virus-specific CD8 + cytotoxic T lymphocytes (CTLs) progressively lose their ability to mount effective antiviral responses. This "exhaustion" is coupled to persistent upregulation of inhibitory receptor programmed death-1 (PD-1) (Pdcd1)-key in suppressing antiviral CTL responses. Here, we investigate allelic Pdcd1 subnuclear localization and transcription during acute and chronic lymphocytic choriomeningitis virus (LCMV) infection in mice. Pdcd1 alleles dissociate from transcriptionally repressive chromatin domains (lamin B) in virus-specific exhausted CTLs but not in naive or effector CTLs. Relative to naive CTLs, nuclear positioning and Pdcd1-lamina dissociation in exhausted CTLs reflect loss of Pdcd1 promoter methylation and greater PD-1 upregulation, although a direct correlation is not observed in effector cells, 8 days post-infection. Genetic deletion of B lymphocyte-induced maturation protein 1 (Blimp-1) enhances Pdcd1-lamina dissociation in effector CTLs, suggesting that Blimp-1 contributes to maintaining Pdcd1 localization to repressive lamina. Our results identify mechanisms governing Pdcd1 subnuclear localization and the broader role of chromatin dynamics in T cell exhaustion., Competing Interests: Declaration of interests Catarina Sacristán is an employee of Cell Press, Elsevier, but was uninformed of manuscript handling. Peer review was fully independent of said author., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Formation of the immunological synapse (IS) is a key event during initiation of an adaptive immune response to a specific antigen. During this process, a T cell and an antigen presenting cell form a stable contact that allows the T cell to integrate both internal and external stimuli in order to decide whether to activate. The threshold for T cell activation depends on the strength and frequency of the calcium (Ca 2+ ) signaling induced by antigen recognition, and it must be tightly regulated to avoid undesired harm to healthy cells. Potassium (K + ) channels are recruited to the IS to maintain the negative membrane potential required to sustain Ca 2+ entry. However, the precise localization of K + channels within the IS remains unknown. Here, we visualized the dynamic subsynaptic distribution of Kv1.3, the main voltage-gated potassium channel in human T cells. Upon T cell receptor engagement, Kv1.3 polarized toward the synaptic cleft and diffused throughout the F-actin rich distal compartment of the synaptic interface-an effect enhanced by CD2-CD58 corolla formation. As the synapse matured, Kv1.3 clusters were internalized at the center of the IS and released in extracellular vesicles. We propose a model in which specific distribution of Kv1.3 within the synapse indirectly regulates the channel function and that this process is limited through Kv1.3 internalization and release in extracellular vesicles., Competing Interests: Declaration of interests Authors declare that they have no competing interests., (Copyright © 2023 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Important signaling events at the immunological synapse have increasingly been linked to cis interactions between receptors on T cells. In this issue of Immunity, Zhao et al. 1 implicate cis CD28/B7 interactions facilitated by curved membrane invaginations in boosting tumor immunity., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Pancreatic ductal adenocarcinoma (PDAC) is among the deadliest types of cancer and has a 5-year survival of less than 8% owing to its complex biology. As PDAC is refractory to immunotherapy, we need to understand the functional dynamics of T cells in the PDAC microenvironment to develop alternative therapeutic strategies. In this study, we performed RNA velocity-based pseudotime analysis on a scRNA-seq dataset from surgically resected human PDAC specimens to gain insight into temporal gene expression patterns that best characterize the cell fates. The tumor microenvironment was seen to encompass a range of terminal states for the T cell trajectories with suppressive and non-tumor-responsive T cells dominating them. However, the results also reveal the existence of a functional branch of the T cell population that was not transitioning to exhausted and senescent states. These findings reveal various microenvironmental signals driving T cell patterns which can be useful in identifying new therapeutic avenues., Competing Interests: The authors have no conflicts of interest to declare based on the scope of this work., (Crown Copyright © 2023.)

Support for basic science has been eclipsed by initiatives aimed at specific medical problems. The latest example is the dismantling of the Skirball Institute at NYU School of Medicine. Here, we reflect on the achievements and mission underlying the Skirball to gain insight into the dividends of maintaining a basic science vision within the academic enterprises., Competing Interests: Declaration of interests Agnel Sfeir is a co-founder, consultant, and shareholder in Repare Therapeutics. Gord Fishell is a co-founder, consultant, and shareholder in Regel Therapeutics. Alexander Schier, Michael Dustin, Wen-Biao Gan, Alexandra Joyner, Ruth Lehmann, David Ron, David Roth, William S. Talbot, Deborah Yelon, and Arturo Zychlinsky declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Small immunoglobulin superfamily (sIGSF) adhesion complexes form a corolla of microdomains around an integrin ring and secretory core during immunological synapse (IS) formation. The corolla recruits and retains major costimulatory/checkpoint complexes, such as CD28, making forces that govern corolla formation of particular interest. Here, we investigated the mechanisms underlying molecular reorganization of CD2, an adhesion and costimulatory molecule of the sIGSF family during IS formation. Computer simulations showed passive distal exclusion of CD2 complexes under weak interactions with the ramified F-actin transport network. Attractive forces between CD2 and CD28 complexes relocate CD28 from the IS center to the corolla. Size-based sorting interactions with large glycocalyx components, such as CD45, or short-range CD2 self-attraction successfully explain the corolla 'petals.' This establishes a general simulation framework for complex pattern formation observed in cell-bilayer and cell-cell interfaces, and the suggestion of new therapeutic targets, where boosting or impairing characteristic pattern formation can be pivotal., Competing Interests: The authors have no competing interests to declare., (© 2021 The Authors.)

The tumor necrosis factor receptor superfamily member HVEM is one of the most frequently mutated surface proteins in germinal center (GC)-derived B cell lymphomas. We found that HVEM deficiency increased B cell competitiveness during pre-GC and GC responses. The immunoglobulin (Ig) superfamily protein BTLA regulated HVEM-expressing B cell responses independently of B-cell-intrinsic signaling via HVEM or BTLA. BTLA signaling into T cells through the phosphatase SHP1 reduced T cell receptor (TCR) signaling and preformed CD40 ligand mobilization to the immunological synapse, thus diminishing the help delivered to B cells. Moreover, T cell deficiency in BTLA cooperated with B cell Bcl-2 overexpression, leading to GC B cell outgrowth. These results establish that HVEM restrains the T helper signals delivered to B cells to influence GC selection outcomes, and they suggest that BTLA functions as a cell-extrinsic suppressor of GC B cell lymphomagenesis., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

One of the 2019 Canada Gairdner International Awards recognizes Timothy Springer's discovery of the first immune system adhesion molecules involved in lymphocyte homing and the translation of those discoveries into therapeutics for autoimmune disease and cancer., (Copyright © 2019. Published by Elsevier Inc.)

Invariant Vγ9Vδ2 T cells respond to "phosphoantigen" metabolites through binding to the B30.2 domain of butyrophilin BTN3A. Yang et al. (2019) use molecular dynamic simulations based on X-ray structures of distinct B30.2 domain dimers to identify the asymmetric dimer as most active, which has implications for the inside-out signaling mechanism., (Copyright © 2019 Elsevier Inc. All rights reserved.)

During immunological synapse (IS) formation, T cell receptor (TCR) signaling complexes, integrins, and costimulatory molecules exhibit a particular spatial localization. Here, we develop an agent-based model for the IS formation based on TCR peptide-bound major histocompatibility complex (pMHC) and leukocyte-function-associated antigen 1 (LFA-1) intracellular activation molecule 1 (ICAM-1) dynamics, including CD28 binding to a costimulatory ligand, coupling of molecules to the centripetal actin flow, and size-based segregation (SBS). A radial gradient of LFA-1 in the peripheral supramolecular activation cluster (pSMAC) toward the central supramolecular activation cluster (cSMAC) emerged as a combined consequence of actin binding and diffusion and modified the positioning of other molecules. The simulations predict a mechanism of CD28 movement, according to which CD28-CD80 complexes passively follow TCR-pMHC microclusters. However, the characteristic CD28-CD80 localization in a ring pattern around the cSMAC only emerges with a particular CD28-actin coupling strength that induces a centripetal motion. These results have implications for the understanding of T cell activation and fate decisions., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

T cells engage in two modes of interaction with antigen-presenting surfaces: stable synapses and motile kinapses. Although it is surmised that durable interactions of T cells with antigen-presenting cells involve synapses, in situ 3D imaging cannot resolve the mode of interaction. We have established in vitro 2D platforms and quantitative metrics to determine cell-intrinsic modes of interaction when T cells are faced with spatially continuous or restricted stimulation. All major resting human T cell subsets, except memory CD8 T cells, spend more time in the kinapse mode on continuous stimulatory surfaces. Surprisingly, we did not observe any concordant relationship between the mode and durability of interaction on cell-sized stimulatory spots. Naive CD8 T cells maintain kinapses for more than 3 hr before leaving stimulatory spots, whereas their memory counterparts maintain synapses for only an hour before leaving. Thus, durable interactions do not require stable synapses., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

Regulatory T (Treg) cell infiltration constitutes a prominent feature of pancreatic ductal adenocarcinoma (PDA). However, the immunomodulatory function of Treg cells in PDA is poorly understood. Here, we demonstrate that Treg cell ablation is sufficient to evoke effective anti-tumor immune response in early and advanced pancreatic tumorigenesis in mice. This response is dependent on interferon-γ (IFN-γ)-producing cytotoxic CD8 + T cells. We show that Treg cells engage in extended interactions with tumor-associated CD11c + dendritic cells (DCs) and restrain their immunogenic function by suppressing the expression of costimulatory ligands necessary for CD8 + T cell activation. Consequently, tumor-associated CD8 + T cells fail to display effector activities when Treg cell ablation is combined with DC depletion. We propose that tumor-infiltrating Treg cells can promote immune tolerance by suppressing tumor-associated DC immunogenicity. The therapeutic manipulation of this axis might provide an effective approach for the targeting of PDA., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

One of the key research areas surrounding HIV-1 concerns the regulation of the fusion event that occurs between the virus particle and the host cell during entry. Even if it is universally accepted that the large GTPase dynamin-2 is important during HIV-1 entry, its exact role during the first steps of HIV-1 infection is not well characterized. Here, we have utilized a multidisciplinary approach to study the DNM2 role during fusion of HIV-1 in primary resting CD4 T and TZM-bl cells. We have combined advanced light microscopy and functional cell-based assays to experimentally assess the role of dynamin-2 during these processes. Overall, our data suggest that dynamin-2, as a tetramer, might help to establish hemi-fusion and stabilizes the pore during HIV-1 fusion., (Copyright © 2017 University of Oxford. Published by Elsevier Inc. All rights reserved.)

Inflammation is paramount in pancreatic oncogenesis. We identified a uniquely activated γδT cell population, which constituted ∼40% of tumor-infiltrating T cells in human pancreatic ductal adenocarcinoma (PDA). Recruitment and activation of γδT cells was contingent on diverse chemokine signals. Deletion, depletion, or blockade of γδT cell recruitment was protective against PDA and resulted in increased infiltration, activation, and Th1 polarization of αβT cells. Although αβT cells were dispensable to outcome in PDA, they became indispensable mediators of tumor protection upon γδT cell ablation. PDA-infiltrating γδT cells expressed high levels of exhaustion ligands and thereby negated adaptive anti-tumor immunity. Blockade of PD-L1 in γδT cells enhanced CD4(+) and CD8(+) T cell infiltration and immunogenicity and induced tumor protection suggesting that γδT cells are critical sources of immune-suppressive checkpoint ligands in PDA. We describe γδT cells as central regulators of effector T cell activation in cancer via novel cross-talk., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Phagocytosis is key for many organismal functions. In a recent issue of Cell, Freeman et al. (2016) demonstrate a feed-forward signaling mechanism wherein F-actin and integrin receptors drive contact formation between phagocytes and antibody-coated solid particles, signaling their engulfment. This mechanism translates nanoscale proximity effects into wider self-propagating signals., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Cells communicate by sensing diffusible or surface-associated chemical signals, with the surface-associated molecules also providing mechanical cues. New work now shows that both chemical and mechanical signals are critical for the communication between T cells and antigen-presenting dendritic cells in the initiation and maintenance of immune responses., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Molecular interactions at the interface between helper T cells and antigen-presenting B cells govern the ability to produce specific antibodies, which is a central event in protective immunity generated by natural infection or man-made vaccines. In order for a T cell to deliver effective help to a B cell and guide affinity maturation, it needs to provide feedback that is proportional to the amount of antigen the B cell collects with its surface antibody. This review focuses on mechanisms by which T and B cells manage to count the products of antigen capture and encourage B cells with the best receptors to dominate the response and make antibody-producing plasma cells. We discuss what is known about the proportionality of T cells responses to presented antigens and consider the mechanisms that B cells may use to keep count of positive feedback from T cells., (Copyright © 2014 Elsevier Inc. All rights reserved.)

T cells are among the most sensitive of cells, but in this issue of Immunity, Honda et al. (2014) demonstrate that effector T cells must lose their touch within hours to protect the host from immunopathology., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Mechanical forces play an increasingly recognized role in modulating cell function. This report demonstrates mechanosensing by T cells, using polyacrylamide gels presenting ligands to CD3 and CD28. Naive CD4 T cells exhibited stronger activation, as measured by attachment and secretion of IL-2, with increasing substrate elastic modulus over the range of 10-200 kPa. By presenting these ligands on different surfaces, this report further demonstrates that mechanosensing is more strongly associated with CD3 rather than CD28 signaling. Finally, phospho-specific staining for Zap70 and Src family kinase proteins suggests that sensing of substrate rigidity occurs at least in part by processes downstream of T-cell receptor activation. The ability of T cells to quantitatively respond to substrate rigidly provides an intriguing new model for mechanobiology., (Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

The germinal center (GC) reaction produces high-affinity antibodies by random mutation and selective clonal expansion of B cells with high-affinity receptors. The mechanism by which B cells are selected remains unclear, as does the role of the two anatomically defined areas of the GC, light zone (LZ) and dark zone (DZ). We combined a transgenic photoactivatable fluorescent protein tracer with multiphoton laser-scanning microscopy and flow cytometry to examine anatomically defined LZ and DZ B cells and GC selection. We find that B cell division is restricted to the DZ, with a net vector of B cell movement from the DZ to the LZ. The decision to return to the DZ and undergo clonal expansion is controlled by T helper cells in the GC LZ, which discern between LZ B cells based on the amount of antigen captured and presented. Thus, T cell help, and not direct competition for antigen, is the limiting factor in GC selection., (Copyright © 2010 Elsevier Inc. All rights reserved.)

In this study, we imaged the differentiation and migratory behavior of nascent plasma cells (PCs) in mouse lymph nodes by intravital microscopy. Pre-PCs exhibited a unique migration pattern characterized by long, linear paths that were randomly oriented. Although chemotaxis via Galphai coupled-receptors has been implicated in PC migration, treatment with Pertussis toxin (Ptx), which ablates these signals, did not prevent movement of pre-PCs while it arrested other lymphocytes. In vitro, pre-PCs displayed processive amoeboid locomotion on surfaces coated with integrin ligand, whereas fully differentiated PCs moved slowly or were arrested. Both PC arrest and differentiation occurred in the medullary cords. Ptx treatment before PC differentiation blocked their accumulation in the medullary cords but pre-PCs still differentiated in other lymph node regions. Taken together, we suggest pre-PCs undergo a persistent random walk to find the medullary cords, where localized chemokines help retain these cells until they undergo differentiation and arrest in situ., (Copyright 2010 Elsevier Inc. All rights reserved.)

Agonist MHC-peptide complexes in the immunological synapse (IS) signal through T cell receptor (TCR) microclusters (MCs) that converge into a central supramolecular activation cluster (cSMAC). The determinants and function of the cSMAC remain unknown. We demonstrate an essential role for ubiquitin (Ub) and TSG101, but less so for HRS, in signal processing events at the cSMAC. Using siRNA in primary T cells, we show that Ub recognition by TSG101 is required for cSMAC formation, TCR MC signal termination, TCR downregulation, and segregation of TCR-MHC-peptide from PKC-theta-enriched signaling complexes. Weak agonist MHC-peptide induced CD80-dependent TCR MCs that dissociated in the center of the IS without recruiting TSG101. These results support TSG101-dependent recognition of CD80-independent TCR MCs as a molecular checkpoint for TCR downregulation., (Copyright 2010 Elsevier Inc. All rights reserved.)

Cytolytic granules mediate killing of virus-infected cells by cytotoxic T lymphocytes. We show here that the granules can take long or short paths to the secretory domain. Both paths utilized the same intracellular molecular events, which have different spatial and temporal arrangements and are regulated by the kinetics of Ca(2+)-mediated signaling. Rapid signaling caused swift granule concentration near the microtubule-organizing center (MTOC) and subsequent delivery by the polarized MTOC directly to the secretory domain-the shortest path. Indolent signaling led to late recruitment of granules that moved along microtubules to the periphery of the synapse and then moved tangentially to fuse at the outer edge of the secretory domain-a longer path. The short pathway is associated with faster granule release and more efficient killing than the long pathway. Thus, the kinetics of early signaling regulates the quality of the T cell cytolytic response.

Cytotoxic lymphocytes kill target cells by releasing the content of secretory lysosomes at the immune synapse. To understand the dynamics and control of cytotoxic immune synapses, we imaged human primary, live natural killer cells on lipid bilayers carrying ligands of activation receptors. Formation of an organized synapse was dependent on the presence of the beta2 integrin ligand ICAM-1. Ligands of coactivation receptors 2B4 and NKG2D segregated into central and peripheral regions, respectively. Lysosomal protein LAMP-1 that was exocytosed during degranulation accumulated in a large and spatially stable cluster, which overlapped with a site of membrane internalization. Lysosomal compartments reached the plasma membrane at focal points adjacent to centrally accumulated LAMP-1. Imaging of fixed cells revealed that perforin-containing granules were juxtaposed to an intracellular compartment where exocytosed LAMP-1 was retrieved. Thus, cytotoxic immune synapses include a central region of bidirectional vesicular traffic, which is controlled by integrin signaling.

Classical alphabeta T cells protect the host by monitoring intracellular and extracellular proteins in a two-step process. The first step is protein degradation and combination with a major histocompatibility complex (MHC) molecule, leading to surface expression of this amalgam (antigen processing). The second step is the interaction of the T cell receptor with the MHC-peptide complex, leading to signaling in the T cells (antigen recognition). The context for this interaction is a T cell-antigen presenting cell junction, known as an immunological synapse if symmetric and stable and as a kinapse if asymmetric and mobile. The physiological recognition of a ligand takes place most efficiently in the F-actin-rich lamellipodium and is F-actin dependent in stages of formation and triggering and myosin II dependent for signal amplification. This review discusses how these concepts emerged from early studies on adhesion, signaling, and cell biology of T cells.

T cell activation is mediated by microclusters (MCs) containing T cell receptors (TCRs), kinases, and adaptors. Although TCR MCs translocate to form a central supramolecular activation cluster (cSMAC) of the immunological synapse at the interface of a T cell and an antigen-presenting cell, the role of MC translocation in T cell signaling remains unclear. Here, we found that the accumulation of MCs at cSMAC was important for T cell costimulation. Costimulatory receptor CD28 was initially recruited coordinately with TCR to MCs, and its signals were mediated through the assembly with the kinase PKCtheta. The accumulation of MCs at the cSMAC was accompanied by the segregation of CD28 from the TCR, which resulted in the translocation of both CD28 and PKCtheta to a spatially unique subregion of cSMAC. Thus, costimulation is mediated by the generation of a unique costimulatory compartment in the cSMAC via the dynamic regulation of MC translocation.

Neuromuscular synapse formation requires a complex exchange of signals between motor neurons and skeletal muscle fibers, leading to the accumulation of postsynaptic proteins, including acetylcholine receptors in the muscle membrane and specialized release sites, or active zones in the presynaptic nerve terminal. MuSK, a receptor tyrosine kinase that is expressed in skeletal muscle, and Agrin, a motor neuron-derived ligand that stimulates MuSK phosphorylation, play critical roles in synaptic differentiation, as synapses do not form in their absence, and mutations in MuSK or downstream effectors are a major cause of a group of neuromuscular disorders, termed congenital myasthenic syndromes (CMS). How Agrin activates MuSK and stimulates synaptic differentiation is not known and remains a fundamental gap in our understanding of signaling at neuromuscular synapses. Here, we report that Lrp4, a member of the LDLR family, is a receptor for Agrin, forms a complex with MuSK, and mediates MuSK activation by Agrin.

The immunological synapse is a stable intercellular structure that specializes in substance and signal transfer from one immune cell to another. Its formation is regulated in part by the diffusion of adhesion and signaling molecules into, and their binding of countermolecules in the contact area. The stability of immunological synapses allows receptor-ligand interactions to approximate chemical equilibrium despite other dynamic aspects. We have developed a mathematical model that describes the coupled reaction-diffusion process in an established immunological synapse. In this study, we extend a previously described contact area fluorescence recovery after photobleaching (FRAP) experiment to test the validity of the model. The receptor binding activity and lateral mobility of fluorescently labeled, lipid-anchored ligands in the bilayer resulted in their accumulation, as revealed by a much higher fluorescence intensity inside the contact area than outside. After complete photobleaching of the synapse, fluorescence recovery requires ligands to dissociate and rebind, and to diffuse in and out of the contact area. Such a FRAP time course consequently provides information on reaction and diffusion, which can be extracted by fitting the model solution to the data. Surprisingly, reverse rates in the two-dimensional contact area were at least 100-fold slower than in three-dimensional solution. As previously reported in immunological synapses, a significant nonrecoverable fraction of fluorescence was observed with one of two systems studied, suggesting some ligands either dissociated or diffused much more slowly compared with other ligands in the same synapse. The combined theory and experiment thus provides a new method for in situ measurements of kinetic rates, diffusion coefficients, and nonrecoverable fractions of interacting molecules in immunological synapses and other stable cell-bilayer junctions.

Kinetic rates and binding affinity of receptor-ligand interactions are important determinants of cell adhesion. Measurements of these parameters in fluid phase using soluble molecules (i.e., three-dimensionial parameters) do not necessarily correlate with their counterparts measured when both binding partners are respectively anchored to two apposing surfaces (i.e., two-dimensional (2D) parameters). Moreover, 2D affinities measured by different methods can differ by orders of magnitude. Here we describe a coupled diffusion-reaction model for the fluorescence recovery after photobleaching experiment previously used to demonstrate the dynamics of adhesive bonds in the contact area. Applying the mathematical model to the contact area fluorescence recovery after photobleaching experiment enables in situ measurements of 2D kinetic rates of the adhesion molecules and their retarded diffusion in a stable contact area. The mathematical properties of the model are characterized in this article and its experimental validation will be presented in the companion article.

Recognition of peptide antigen by T cells involves coordinated movement of T cell receptors (TCRs) along with other costimulatory and signaling molecules. The spatially organized configurations that result are collectively referred to as the immunological synapse. Experimental investigation of the role of spatial organization in TCR signaling has been facilitated by the use of nanopatterned-supported membranes to direct TCR into alternative patterns. Here we study the mechanism by which substrate structures redirect TCR transport. Using a flow-tracking algorithm, the ensemble of TCR clusters within each cell was tracked during synapse formation under various constraint geometries. Shortly after initial cluster formation, a coordinated centripetal flow of approximately 20 nm/s develops. Clusters that encounter substrate-imposed constraint are deflected and move parallel to the constraint at speeds that scale with the relative angle of motion to the preferred centripetal direction. TCR transport is driven by actin polymerization, and the distribution of F-actin was imaged at various time points during the synapse formation process. At early time points, there is no significant effect on actin distribution produced by substrate constraints. At later time points, modest differences were observed. These data are consistent with a frictional model of TCR coupling to cytoskeletal flow, which allows slip. Implications of this model regarding spatial sorting of cell-surface molecules are discussed.

Cell polarity is critical for T lymphocyte movement during their hunt for antigen-bearing cells and for infected target cells. In this issue of Cell, Yeh et al. (2008) now reveal a direct link between T cell polarity and the production of proinflammatory cytokines in mice lacking the class I MHC-restricted T cell-associated molecule (Crtam).

The lipid sphingosine-1-phosphate has been identified as a key exit signal for lymph nodes. In this issue of Immunity, Pham et al. (2008) show that its action can only be understood in the context of retention signals transduced by CCR7.

The immunological synapse (IS) is a junction between the T cell and antigen-presenting cell and is composed of supramolecular activation clusters (SMACs). No studies have been published on naive T cell IS dynamics. Here, we find that IS formation during antigen recognition comprises cycles of stable IS formation and autonomous naive T cell migration. The migration phase is driven by PKCtheta, which is localized to the F-actin-dependent peripheral (p)SMAC. PKCtheta(-/-) T cells formed hyperstable IS in vitro and in vivo and, like WT cells, displayed fast oscillations in the distal SMAC, but they showed reduced slow oscillations in pSMAC integrity. IS reformation is driven by the Wiscott Aldrich Syndrome protein (WASp). WASp(-/-) T cells displayed normal IS formation but were unable to reform IS after migration unless PKCtheta was inhibited. Thus, opposing effects of PKCtheta and WASp control IS stability through pSMAC symmetry breaking and reformation.

We formulate a general analysis to determine the two-dimensional dissociation constant (2D Kd), and use this method to study the interaction of CD2-expressing T cells with glass-supported planar bilayers containing fluorescently labeled CD58, a CD2 counter-receptor. Both CD2 and CD58 are laterally mobile in their respective membranes. Adhesion is indicated by accumulation of CD2 and CD58 in the cell-bilayer contact area; adhesion molecule density and contact area size attain equilibrium within 40 min. The standard (Scatchard) analysis of solution-phase binding is not applicable to the case of laterally mobile adhesion molecules due to the dynamic nature of the interaction. We derive a new binding equation, B/F=[(Ntxf)/(KdxScell)]-[(Bxp)/Kd], where B and F are bound and free CD58 density in the contact area, respectively; Nt is CD2 molecule number per cell; f is CD2 fractional mobility; Scell is cell surface area; and p is the ratio of contact area at equilibrium to Scell. We use this analysis to determine that the 2D Kd for CD2-CD58 is 5.4-7.6 molecules/microm2. 2D Kd analysis provides a general and quantitative measure of the mechanisms regulating cell-cell adhesion.

Integrins comprise a large family of cell-cell and cell-matrix adhesion receptors that rapidly modulate their adhesiveness. The arrest of leukocyte integrins on target vascular beds involves instantaneous conformational switches generating shear-resistant adhesions. Structural data suggest that these integrins are maintained in low-affinity conformations and must rapidly undergo conformational switches transduced via cytoplasmic changes ("inside-out" signaling) and simultaneous ligand-induced rearrangements ("outside-in"). This bidirectional activation is accelerated by signals from endothelial chemoattractants (chemokines). Recent studies predict that shear forces in the piconewton (pN) range per integrin can facilitate these biochemical switches. After extravasation, antigen recognition involves smaller internal forces from cytoskeletal motors and actin polymers forming the immune synapse. In this review, we address how forces facilitate allosteric integrin activation by biochemical signals. Evidence suggests that preformed cytoskeletal anchorage rather than free integrin mobility is key for force-enhanced integrin activation by chemokines and TCR signals.

T cell receptor (TCR) signaling is initiated and sustained in microclusters; however, it's not known whether signaling also occurs in the TCR-rich central supramolecular activation cluster (cSMAC). We showed that the cSMAC formed by fusion of microclusters contained more CD45 than microclusters and is a site enriched in lysobisphosphatidic acid, a lipid involved in sorting ubiquitinated membrane proteins for degradation. Calcium signaling via TCR was blocked within 2 min by anti-MHCp treatment and 1 min by latrunculin-A treatment. TCR-MHCp interactions in the cSMAC survived these perturbations for 10 min and hence were not sufficient to sustain signaling. TCR microclusters were also resistant to disruption by anti-MHCp and latrunculin-A treatments. We propose that TCR signaling is sustained by stabilized microclusters and is terminated in the cSMAC, a structure from which TCR are sorted for degradation. Our studies reveal a role for F-actin in TCR signaling beyond microcluster formation.

Adaptive immune responses are initiated by interactions of T cells with antigen-presenting cells, but the basic nature of these interactions during an immune response in vivo has been a matter of speculation. While some in vitro systems provide evidence for stable interactions, referred to as immunological synapses, compelling evidence supports T cell activation through serial transient interactions. Deep tissue intravital and organ culture microscopy studies suggest that both modes of interaction are employed, but new issues have emerged. This review will discuss in vitro results that framed the hypotheses that are currently being tested in vivo. I present a model in which TCR stop signals compete with chemokine-mediated go signals to adjust the duration of immunological synapse formation and tune the immune response between tolerance and full activation.

The integrin LFA-1 and its ligand ICAM-1 mediate B cell adhesion, but their role in membrane-bound antigen recognition is still unknown. Here, using planar lipid bilayers and cells expressing ICAM-1 fused to green fluorescence protein, we found that the engagement of B cell receptor (BCR) promotes B cell adhesion by an LFA-1-mediated mechanism. LFA-1 is recruited to form a mature B cell synapse segregating into a ring around the BCR. This distribution is maintained over a wide range of BCR/antigen affinities (10(6) M(-1) to 10(11) M(-1)). Furthermore, the LFA-1 binding to ICAM-1 reduces the level of antigen required to form the synapse and trigger a B cell. Thus, LFA-1/ICAM-1 interaction lowers the threshold for B cell activation by promoting B cell adhesion and synapse formation.

The immunological synapse formed during mature T cell activation consists of a central cluster of TCR and MHC molecules surrounded by a ring of LFA-1 and ICAM-1. We examined synapse formation in thymocytes undergoing activation in a lipid bilayer system by following the movement of fluorescent MHC and ICAM-1 molecules. Immature CD4(+)CD8(+) thymocytes formed a decentralized synapse with multiple foci of MHC accumulation corresponding to areas of exclusion of ICAM-1. The MHC clusters and ICAM-1 holes were mobile and transient and correlated with active and sustained signaling, as shown by staining with antibodies against phosphotyrosine and activated Lck. Our findings show that signaling in immature thymocytes can result from a novel, multifocal pattern of receptor accumulation.

WIP stabilizes actin filaments and is important for filopodium formation. To define the role of WIP in immunity, we generated WIP-deficient mice. WIP(minus sign/minus sign) mice have normal lymphocyte development, but their T cells fail to proliferate, secrete IL-2, increase their F-actin content, polarize and extend protrusions following T cell receptor ligation, and are deficient in conjugate formation with superantigen-presenting B cells and anti-CD3 bilayers. In contrast, WIP-deficient B lymphocytes have enhanced proliferation and CD69 expression following B cell receptor ligation and mount normal antibody responses to T-independent antigens. Both WIP-deficient T and B cells show a profound defect in their subcortical actin filament networks. These results suggest that WIP is important for immunologic synapse formation and T cell activation.

Recognition of antigen by T cells requires the formation of a specialized junction between the T cell and the antigen-presenting cell. This junction is generated by the recruitment and the exclusion of specific proteins from the contact area. The mechanisms that regulate these events are unknown. Here we demonstrate that ligand engagement of the adhesion molecule, CD2, initiates a process of protein segregation, CD2 clustering, and cytoskeletal polarization. Although protein segregation was not dependent on the cytoplasmic domain of CD2, CD2 clustering and cytoskeletal polarization required an interaction of the CD2 cytoplasmic domain with a novel SH3-containing protein. This novel protein, called CD2AP, is likely to facilitate receptor patterning in the contact area by linking specific adhesion receptors to the cytoskeleton.